Major Sphingolipid Research Articles

The ORP9-ORP11 dimer promotes sphingomyelin synthesis

Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.

The ORP9-ORP11 dimer promotes sphingomyelin synthesis.

Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.

A non-toxic equinatoxin-II reveals the dynamics and distribution of sphingomyelin in the cytosolic leaflet of the plasma membrane

Sphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, single molecule observation at high resolutions down to 1 ms revealed that all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs.

Transcriptomic and Lipidomic Analysis Reveals Complex Regulation Mechanisms Underlying Rice Roots' Response to Salt Stress.

Rice (Oryza sativa L.), a crucial food crop that sustains over half the world's population, is often hindered by salt stress during various growth stages, ultimately causing a decrease in yield. However, the specific mechanism of rice roots' response to salt stress remains largely unknown. In this study, transcriptomics and lipidomics were used to analyze the changes in the lipid metabolism and gene expression profiles of rice roots in response to salt stress. The results showed that salt stress significantly inhibited rice roots' growth and increased the roots' MDA content. Furthermore, 1286 differentially expressed genes including 526 upregulated and 760 downregulated, were identified as responding to salt stress in rice roots. The lipidomic analysis revealed that the composition and unsaturation of membrane lipids were significantly altered. In total, 249 lipid molecules were differentially accumulated in rice roots as a response to salt stress. And most of the major phospholipids, such as phosphatidic acid (PA), phosphatidylcholine (PC), and phosphatidylserine (PS), as well as major sphingolipids including ceramide (Cer), phytoceramide (CerP), monohexose ceramide (Hex1Cer), and sphingosine (SPH), were significantly increased, while the triglyceride (TG) molecules decreased. These results suggested that rice roots mitigate salt stress by altering the fluidity and integrity of cell membranes. This study enhances our comprehension of salt stress, offering valuable insights into changes in the lipids and adaptive lipid remodeling in rice's response to salt stress.

Bioaccessibility of Glucosylceramide in Rice Based on the Cooking Condition and Cultivar.

Glucosylceramide (GlcCer), a major sphingolipid in plants, possesses various food functions, including improvement of intestinal impairments. This study evaluated rice cooking conditions and cultivars based on GlcCer levels transferred into the digestive juice using an in vitro digestion model to investigate the factors related to GlcCer availability. GlcCer levels transferred into the digestive juice were higher in rice gruel than in boiled rice. The GlcCer levels in the digestive juice of boiled rice varied based on the rice cultivar, whereas those in rice gruel had no difference. Thus, GlcCer in rice was not fully utilized via digestion. Further, bioaccessibility was related to the amylose ratio and added water content.

Sphingomyelinase Regulates the Release of Protumoral Mitochondria Containing-Vesicles from AML Cells

Mitochondrial transfer from bone marrow stromal cells to acute myeloid leukaemia (AML) blasts is protumoral (Marlein et al., 2017; Moschoi et al., 2016) . We have previously shown that this was through a NADPH oxidase-2 (Nox2) dependent mechanism which increases AML fitness both in vitro and in vivo. Furthermore, we recently have shown that AML cells export mitochondria in vesicles which are subsequently phagocytosed by bone marrow macrophages (Moore et al., 2022) . Next, we have investigated how the release of mitochondria-containing vesicles is regulated and how this process contributes to the fitness of AML blasts. Sphingolipids are a component of the cell membrane which impact the stability and curvature of the membrane. They have been shown to play a role in the biogenesis of vesicles and a number of the enzymes involved in sphingolipid metabolism are dysregulated in AML, therefore we investigated the role of sphingolipids in mitochondrial exocytosis via vesicle formation. To understand the role of vesicles in regulating AML fitness, we used primary human AML (collected with approval on the East of England - Cambridgeshire and Hertfordshire Research Ethics Committee and the UK Health Research Authority, IRAS Project ID - 33753) and mouse syngeneic AML models (MN1 and HOXA9/Meis). We find that the myosin inhibitor blebbistatin inhibits vesicle release and results in an increased mitochondrial mass and increased apoptosis. In order to investigate the involvement of one of the major sphingolipids, ceramide, we targeted neutral sphingomyelinase (GW4869) which is responsible for ceramide synthesis. We treated the AML cells in vitro with GW4869 for 24 h which induced a large increase in vesicle release. Using the mitochondrial stain Mitotracker Green (MTG) we confirm that these vesicles contain mitochondria. The vesicles were isolated, DNA was extracted, and RT-qPCR performed which confirms increased mitochondrial DNA content. Others have previously shown that low levels of cellular reactive oxygen species (ROS) are associated with leukaemic stem cells (LSCs). DCF staining of AML treated with GW4869 shows low levels of cellular ROS. To assess how this process might impact the stemness of AML, we performed in vivo experiments. Pretreatment of MN1 cells with GW4869 accelerated engraftment in C57Bl/6 animals which corresponded with a decrease in cellular ROS. In summary, we have demonstrated the mechanism of mitochondrial exocytosis is important for AML fitness and that by disrupting sphingolipid metabolism, dysregulation of this process leads to increased tumour burden.

Plasma Sphingolipid Profile of Healthy Black and White Adults Differs Based on Their Parental History of Type 2 Diabetes.

Ceramides and sphingolipids have been linked to type 2 diabetes (T2D). The Ceramides and Sphingolipids as Predictors of Incident Dysglycemia (CASPID) study is designed to determine the association of plasma sphingolipids with the pathophysiology of human T2D. A comparison of plasma sphingolipids profiles in Black and White adults with (FH+) and without (FH-) family history of T2D. We recruited 100 Black and White FH- (54 Black, 46 White) and 140 FH+ (75 Black, 65 White) adults. Fasting plasma levels of 58 sphingolipid species, including 18 each from 3 major classes (ceramides, monohexosylceramides, and sphingomyelins, all with 18:1 sphingoid base) and 4 long-chain sphingoid base-containing species, were measured by liquid chromatography/mass spectrometry. Sphingomyelin was the most abundant sphingolipid in plasma (89% in FH-), and was significantly elevated in FH+ subjects (93%). Ceramides and monohexosylceramides comprised 5% and 6% of total sphingolipids in the plasma of FH- subjects, and were reduced significantly in FH+ subjects (3% and 4%, respectively). In FH+ subjects, most ceramide and monohexosylceramide species were decreased but sphingomyelin species were increased. The level of C18:1 species of all 3 classes was elevated in FH+ subjects. Elevated levels of sphingomyelin, the major sphingolipids of plasma, and oleic acid-containing sphingolipids in healthy FH+ subjects compared with healthy FH- subjects may reflect heritable elements linking sphingolipids and the development of T2D.

Quercetin Affects Ceramide but not Sphingosine-1-phosphate in ras-transformed 3T3 Fibroblasts

Investigations were carried out on the effect of quercetin on the level of the major sphingolipid metabolites in ras-transformed 3T3 fibroblasts. Quercetin is a polyphenol of a significant biomedical importance, which has been reported to show beneficial effects such as antioxidant, anti-neoplastic, anti-ageing, etc. Sphingolipids are functionally active lipid molecules, which regulate important cellular processes, like proliferation, apoptosis and transmembrane signalling, among others. Despite the numerous investigations devoted to the influence of quercetin on the lipid metabolism of various types of cancer cells, the mechanism of this influence on the major sphingolipid pathways still remains unclear. Our studies showed that sphingomyelin was decreased in ras-transformed 3T3 fibroblasts, whereas ceramide, which is a pro-apoptotic factor, was increased as a result of elevated neutral sphingomyelinase activity. However, the level of sphingosine-1-phosphate, which is related to cell proliferation, was not altered as a result of quercetin treatment, which is an unexpected finding when compared to our previous studies with cancer cells. In addition, the observed changes in the balancing enzyme in the sphingolipid pathway, sphingosine kinase 1, which produces sphingosine-1-phosphate, were not statistically significant in quercetin-treated oncogene-transformed fibroblasts. The presented data are useful for better understanding of the effect of flavonoids on the regulation of sphingolipid metabolism and could help in the development of complex antitumour therapeutic approaches, involving natural antitumour agents like polyphenols.

Abstract 1585: Targeting acid ceramidase-1 to inhibit sphingolipid metabolism and tumor growth in pancreatic ductal carcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths in the United States. PDAC is an aggressive disease with a poor survival rate of <10%. Hence, there is an urgent need to find novel drug targets for the clinical management of PDAC. Altered sphingolipid metabolism (SM) is a hallmark of cancer. The major sphingolipid metabolites are ceramide, sphingosine and sphingosine-1-phosphate (S1P). We found that S1P is the predominant SM metabolite in PDAC cells and tissues. The equilibrium between ceramide and S1P levels within the body is an important factor to determine cell fate. Ceramide induces apoptosis, while S1P promotes cancer growth. The IHC and ELISA assays showed that S1P levels are higher in PDAC cells and tissues compared to normal pancreatic tissue. We found that S1P induces spheroid formation in PDAC cells at nanomolar concentrations when compared to C1P and sphingosine. S1P binds to S1P receptors (SIPR1-5) to activate downstream signaling. Mechanistically, S1P treatment activates beta-catenin and notch signaling pathway proteins in PDAC cells. Our data suggest that S1P promotes tumor growth and stemness in PDAC and inhibition of S1P production can be a novel strategy for PDAC treatment. The cancer genome atlas (TCGA) data analysis indicated that genes involved in S1P production, especially acid ceramidase (ASAH1) and sphingosine kinase 1 (SPHK1) are upregulated in PDAC. ASAH1 converts ceramide to sphingosine which further gets converted to S1P by SPHK1. SPHK1 has been targeted in cancers, but kinase inhibitors are traditionally known for their non-selectivity and toxicity. Moreover, selective targeting of S1PR1-5 has been unsuccessful. Hence, there is a need to identify other targets to inhibit the production of S1P. Therefore, we decided to target Acid ceramidase 1 (ASAH1). Inhibiting ASAH1 will result in the accumulation of proapoptotic ceramide and inhibition of S1P production. Our data showed that ASAH1 is overexpressed in PDAC tissue and cells, while the ASAH1 knockdown in PDAC cells resulted in reduced proliferation, colony formation, migration and spheroid formation. Hence, targeting ASAH1 represents a novel strategy to inhibit PDAC progression by inhibiting sphingolipid metabolism and S1P production. Next, we developed a series of ASAH1 inhibitors based on Cerenib-2 (known ASAH1 inhibitor). One of the analogs, Cer-IVA inhibited the proliferation, colony and spheroid formation in PDAC cells at nanomolar concentration. Moreover, it also induced apoptosis and G2/M-cell cycle arrest in a dose and time-dependent manner. Further, it inhibited S1P production and increased ceramide production in PDAC cells. We are currently investigating the antitumor effects of Cer-IVA in an orthotopic model in C57BL/6 mice. In conclusion, targeting ASAH1 by Cer-IVA is an attractive strategy to inhibit S1P production, growth and stemness in PDAC cells. Citation Format: Krishan G. Jain, Hindole Ghosh, Sangita Bhattacharyya, Anup Kasi, Ameer Hamza, Scott J. Weir, Michael VanSaun, Stefan Bossmann, Shrikant Anant, Bernhard Biersack, Prasad R. Dandawate. Targeting acid ceramidase-1 to inhibit sphingolipid metabolism and tumor growth in pancreatic ductal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1585.

Overexpression of a GIPC glycosyltransferase gene, OsGMT1, suppresses plant immunity and delays heading time in rice

Glycosylinositol phosphorylceramides (GIPCs) are the major sphingolipids in the plant plasma membrane. In Arabidopsis, mutations of genes involved in the synthesis of GIPCs affect many physiological aspects of plants, including growth, pollen fertility, defense, and stress signaling. Loss of function of the GIPC MANNOSYL-TRANSFERASE1 (AtGMT1) results in GIPC misglycosylation and induces plant immune responses accompanied by a severely dwarfed phenotype, thus indicating that GIPCs play important roles in plant immunity. Here, we investigated the enzymatic activity and phenotypes of transgenic lines of OsGMT1, the ortholog of AtGMT1. Sphingolipidomic analysis indicated that OsGMT1 retained the enzymatic activity of GIPC hexose (Hex) glycosylation, but the knockout lines did not accumulate H2O2. In contrast, the OsGMT1 overexpression lines showed significant down-regulation of several defense-associated or cell wall synthesis-associated genes, and enhanced sensitivity to rice blast. Furthermore, we first demonstrated the sensitivity of rice cells to MoNLP1 protein through calcein AM release assays using rice protoplasts, thus legitimizing the presence of MoNLPs in rice blast fungus. In addition, yeast two-hybrid screens using OsGMT1 as bait revealed that OsGMT1 may regulate heading time through the OsHAP5C signaling pathway. Together, our findings suggested clear physiological functional differentiation of GMT1 orthologs between rice and Arabidopsis.

Nonspecific phospholipase C3 of radish has phospholipase D activity towards glycosylinositol phosphoceramide.

Glycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots. Peptide mass fingerprinting analysis revealed that the potential candidate for GIPC-PLD protein was nonspecific phospholipase C3 (NPC3), which has not been characterized as a PLD. The recombinant NPC3 protein obtained by heterologous expression system in Escherichia coli produced PC1P from GIPC and showed essentially the same enzymatic properties as those we characterized as GIPC-PLD in cabbage, radish and Arabidopsis thaliana. From these results, we conclude that NPC3 is one of the enzymes that degrade GIPC.

Resveratrol Alters Sphingolipid Metabolism in ras-transformed 3T3 Fibroblasts

The effect of resveratrol on the sphingolipid metabolism has been investigated in ras-transformed 3T3 fibroblasts. Resveratrol is a phytoalexin of a great medico-biological significance, which has been reported to exhibit significant beneficial effects such as antioxidant, lipid-lowering, anti-neoplastic, etc. Sphingolipids are functionally active lipid molecules, which are implicated in important cellular processes, such as proliferation, migration, apoptosis and transmembrane signalling, among others. Although there are many investigations devoted to the influence of resveratrol on the lipid metabolism of various types of cancer cells, the mechanism of this effect on the sphingolipid pathway remains unclear. The present studies showed that the major sphingolipid, sphingomyelin, was decreased in membranes from resveratrol-treated ras-transformed 3T3 fibroblasts, whereas ceramide, which is a pro-apoptotic factor, was elevated due to neutral sphingomyelinase up-regulation. In addition, the level of sphingosine, a product of ceramidase, was increased and the content of sphingosine-1-phosphate, which is related to cell proliferation, was lowered due to resveratrol treatment, which is an important finding especially for cancer cells. The balancing enzyme in the sphingolipid pathway, sphingosine kinase, which produces sphingosine-1-phosphate, was down-regulated in resveratrol-treated oncogene-transformed fibroblasts. In conclusion, the present results clearly show that treatment of ras-transformed fibroblasts with resveratrol induces changes in the major representatives of the sphingolipid metabolic pathway shifting the “sphingolipid rheostat” towards prevalence of the proapoptotic factor ceramide. The reported observations can be applied in the development of complex anti-tumour therapeutic strategies.

Inositol phosphorylceramide synthase null Leishmania are viable and virulent in animal infections where salvage of host sphingomyelin predominates

Many pathogens synthesize inositol phosphorylceramide (IPC) as the major sphingolipid (SL), differing from the mammalian host where sphingomyelin (SM) or more complex SLs predominate. The divergence between IPC synthase and mammalian SL synthases has prompted interest as a potential drug target. However, in the trypanosomatid protozoan Leishmania, cultured insect stage promastigotes lack de novo SL synthesis (Δspt2-) and SLs survive and remain virulent, as infective amastigotes salvage host SLs and continue to produce IPC. To further understand the role of IPC, we generated null IPCS mutants in Leishmania major (Δipcs-). Unexpectedly and unlike fungi where IPCS is essential, Δipcs- was remarkably normal in culture and highly virulent in mouse infections. Both IPCS activity and IPC were absent in Δipcs- promastigotes and amastigotes, arguing against an alternative route of IPC synthesis. Notably, salvaged mammalian SM was highly abundant in purified amastigotes from both WT and Δipcs-, and salvaged SLs could be further metabolized into IPC. SM was about 7-fold more abundant than IPC in WT amastigotes, establishing that SM is the dominant amastigote SL, thereby rendering IPC partially redundant. These data suggest that SM salvage likely plays key roles in the survival and virulence of both WT and Δipcs- parasites in the infected host, confirmation of which will require the development of methods or mutants deficient in host SL/SM uptake in the future. Our findings call into question the suitability of IPCS as a target for chemotherapy, instead suggesting that approaches targeting SM/SL uptake or catabolism may warrant further emphasis.

Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians

Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action. Bryoporin is a protein from the moss Physcomitrium patens, and its corresponding gene was found to be upregulated by various abiotic stresses, especially dehydration, as well as upon fungal infection. Based on the amino acid sequence, it was suggested that bryoporin was related to the actinoporin family of pore-forming proteins, originally discovered in sea anemones. Here, we provide the first detailed structural and functional analysis of this plant cytolysin. The crystal structure of monomeric bryoporin is highly similar to those of actinoporins. Our cryo-EM analysis of its pores showed an actinoporin-like octameric structure, thereby revealing a close kinship of proteins from evolutionarily distant organisms. This was further confirmed by our observation of bryoporin’s preferential binding to and formation of pores in membranes containing animal sphingolipids, such as sphingomyelin and ceramide phosphoethanolamine; however, its binding affinity was weaker than that of actinoporin equinatoxin II. We determined bryoporin did not bind to major sphingolipids found in fungi or plants, and its membrane-binding and pore-forming activity was enhanced by various sterols. Our results suggest that bryoporin could represent a part of the moss defense arsenal, acting as a pore-forming toxin against membranes of potential animal pathogens, parasites, or predators.

Platelet Lipidome Fingerprint: New Assistance to Characterize Platelet Dysfunction in Obesity.

Obesity is associated with a pro-inflammatory and pro-thrombotic state that supports atherosclerosis progression and platelet hyper-reactivity. During the last decade, the platelet lipidome has been considered a treasure trove, as it is a source of biomarkers for preventing and treating different pathologies. The goal of the present study was to determine the lipid profile of platelets from non-diabetic, severely obese patients compared with their age- and sex-matched lean controls. Lipids from washed platelets were isolated and major phospholipids, sphingolipids and neutral lipids were analyzed either by gas chromatography or by liquid chromatography coupled to mass spectrometry. Despite a significant increase in obese patient’s plasma triglycerides, there were no significant differences in the levels of triglycerides in platelets among the two groups. In contrast, total platelet cholesterol was significantly decreased in the obese group. The profiling of phospholipids showed that phosphatidylcholine and phosphatidylethanolamine contents were significantly reduced in platelets from obese patients. On the other hand, no significant differences were found in the sphingomyelin and ceramide levels, although there was also a tendency for reduced levels in the obese group. The outline of the glycerophospholipid and sphingolipid molecular species (fatty-acyl profiles) was similar in the two groups. In summary, these lipidomics data indicate that platelets from obese patients have a unique lipid fingerprint that may guide further studies and provide mechanistic-driven perspectives related to the hyperactivate state of platelets in obesity.

Virtual Screening Approach to Repurpose FDA‐approved Drugs for Targeting Acid Ceramidase‐1 in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer‐related deaths in the US. Despite extensive research and advances in molecular pathogenesis, PDAC remains a major problem in the US. It is also projected to be the second leading cause of cancer‐related deaths in the US by 2030. PDAC is an aggressive disease and characterized by poor prognosis, rapid progression, drug resistance and recurrence. The current chemotherapy treatment only improved survival to <10%, hence there is a need to identify a novel target for drug discovery.Recently, altered sphingolipid metabolism was identified in PDAC patients. Sphingolipids are critical constituents of the cell membrane that regulate several signaling pathways related to cell survival. The major sphingolipid metabolites are ceramide and sphingosine which are interconvertible, while ceramide is pro‐apoptotic and sphingosine is anti‐apoptotic. Ceramidases are a heterogeneous enzyme family that convert ceramides into sphingosine and fatty acids. The five human ceramidases are identified are the acid ceramidase (ASAH1); neutral ceramidase (ASAH2); alkaline ceramidase 1 (ACER1); alkaline ceramidase 2 (ACER2); and alkaline ceramidase 3 (ACER3). To understand the role of ceramides in PDAC, we first interrogated the cancer genome atlas database and found that all ceramides are expressed in lower levels in PDAC except ASAH1 which is uniquely overexpressed in PDAC patients compared to normal individuals. Further, immunocytochemistry of PDAC patient tissue microarray showed higher expression of ASAH1 in PDAC tumors compared to normal pancreas. Western blot analysis also showed overexpression of ASAH1 protein in PDAC cell lines. Studies indicate that ASAH1 plays an important role in PDAC progression. Our data indicate that ASAH1 is a potential target for the treatment of PDAC. For faster translation of this target to the clinic, we used virtual screening and molecular docking approach to identify FDA‐approved drugs for targeting ASAH1. We have downloaded the 3D‐protein structure of ASAH1 from the protein data bank and currently screening a library of small molecular drugs against ASAH1 protein using multiple software programs such as Autodock vina and PyRx. We will select the top 10 drug candidates from virtual screening based on their binding energies and the ability to form hydrogen bonds with key amino acids. We will confirm these findings using a cellular thermal shift assay to analyze the binding of these drugs to ASAH1 in PDAC cell lines and test their anticancer activities in cell culture system and orthotopic PDAC mouse model.

NMMHC IIA triggered lipid metabolize reprogramming resulting in vascular endothelial cellular tight junction injury.

Nonmuscle myosin heavy chain IIA, played an essential role in the promotion of tight junction injury in vascular endothelial cells under oxygen glucose deprivation condition. Rat microvascular endothelial cells had been confirmed to have the susceptibility to ox-LDL stimulation under OGD condition. We proposed the hypothesis that lipid metabolic reprogramming might be the root cause for damage to RBMCs tight junction. Untargeted shotgun and targeted lipid metabolomics mass spectrometry approaches combined with principal component analysis was applied to better define the lipids contributing to the variance observed between control and different OGD time. The protein expression of tight junction of RBMCs: occludin, claudin-5, and ZO-1 were detected with immunofluorescence staining and western blot. The proof of the interaction between NMMHC IIA and SREBP1 was investigated via GST-pull down, while their specific binding fragments were also confirmed. The regulation mechanism of NMMHC IIA on SREBP1 was investigated to explore downstream regulatory signaling pathways. Untargeted and targeted shotgun lipidomics data revealed that OGD might be the conditional factor in reshaping lipid components. Mechanistic studies showed that with the increase of OGD time, PCA analysis of lipidomics obtained from RBMCs indicated their specificity in reshaping lipid components, while ≥80% major lipid components phospholipids and sphingolipids transferred from phospholipids, sphingolipids, and neutral lipids, of which neutrallipids taken the largest proportion with OGD time course. Perturbing reprogramming of lipid composition was less susceptible to OGD condition via knockdown of NMMHC IIA of vascular endothelial cells. Knockdown of NMMHC IIA could promote tight junction defense to OGD condition. NMMHC IIA could directly bind with SREBP1, then could affect sterol regulatory element binding protein-1 to adjust lipid metabolize reprogramming of RBMCs. Mechanistic studies showed that perturbing reprogramming of lipid composition could enhance tight junction damage, which was mediated by the opposing effects of NMMHC IIA.

The major plant sphingolipid long chain base phytosphingosine inhibits growth of bacterial and fungal plant pathogens

Sphingolipid long chain bases (LCBs) are building blocks of sphingolipids and can serve as signalling molecules, but also have antimicrobial activity and were effective in reducing growth of a range of human pathogens. In plants, LCBs are linked to cell death processes and the regulation of defence reactions against pathogens, but their role in directly influencing growth of plant-interacting microorganisms has received little attention. Therefore, we tested the major plant LCB phytosphingosine in in vitro tests with the plant pathogenic fungi Verticillium longisporum, Fusarium graminearum and Sclerotinia sclerotiorum, the plant symbiotic fungal endophyte Serendipita indica, the bacterial pathogens Pseudomonas syringae pv. tomato (Pst), Agrobacterium tumefaciens, and the related beneficial strain Rhizobium radiobacter. Phytosphingosine inhibited growth of these organisms at micromolar concentrations. Among the fungal pathogens, S. sclerotiorum was the most, and F. graminearum was the least sensitive. 15.9 μg/mL phytosphingosine effectively killed 95% of the three bacterial species. Plant disease symptoms and growth of Pst were also inhibited by phytosphingosine when co-infiltrated into Arabidopsis leaves, with no visible negative effect on host tissue. Taken together, we demonstrate that the plant LCB phytosphingosine inhibits growth of plant-interacting microorganisms. We discuss the potential of elevated LCB levels to enhance plant pathogen resistance.

Sphingolipids and Cholesterol.

Sphingolipids and cholesterol are two lipid partners on cellular membranes where they form specific microdomains, named lipid rafts, which mediate specific cell functions. Sphingomyelin (SM) is one of the major sphingolipids. SM and free cholesterol are also two key lipids on the monolayer of plasma lipoproteins, including chylomicron, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), which participate in lipid transport in the circulation. Thus, sphingolipids and cholesterol play a fundamental role in cell membrane structure and blood lipid transport. In this chapter we will discuss the relationship between both lipids, on the cell membrane and in the circulation, as well as the impact of such relationship in the development of metabolic diseases.

Association of Alpha-Crystallin with Fiber Cell Plasma Membrane of the Eye Lens Accompanied by Light Scattering and Cataract Formation.

α-crystallin is a major protein found in the mammalian eye lens that works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress in the eye lens. These functions of α-crystallin are significant for maintaining lens transparency. However, with age and cataract formation, the concentration of α-crystallin in the eye lens cytoplasm decreases with a corresponding increase in the membrane-bound α-crystallin, accompanied by increased light scattering. The purpose of this review is to summarize previous and recent findings of the role of the: (1) lens membrane components, i.e., the major phospholipids (PLs) and sphingolipids, cholesterol (Chol), cholesterol bilayer domains (CBDs), and the integral membrane proteins aquaporin-0 (AQP0; formally MIP26) and connexins, and (2) α-crystallin mutations and post-translational modifications (PTMs) in the association of α-crystallin to the eye lens’s fiber cell plasma membrane, providing thorough insights into a molecular basis of such an association. Furthermore, this review highlights the current knowledge and need for further studies to understand the fundamental molecular processes involved in the association of α-crystallin to the lens membrane, potentially leading to new avenues for preventing cataract formation and progression.